Quantitation of phosphatidylethanol in dried blood after volumetric absorptive microsampling

Stimulated by the increased recognition of phosphatidylethanol (PEth) as sensitive direct marker of alcohol intake, the Ghent University's Laboratory of Toxicology and the National Institute of Criminalistics and Criminology combined their efforts to develop a quantitative method. To facilitate implementation the focus was on the use of a sampling technique which allows quick and easy blood collection, without the need of dedicated personnel at any place/any time. In the meantime the cooperation of the two labs should also allow to initiate a Belgian network of laboratories capable of quantifying PEth.

Dried blood microsamples were collected via volumetric absorptive microsampling (VAMS). PEth 16:0/18:1 was quantified after liquid-liquid extraction using two independent isotope dilution - liquid chromatography - tandem mass spectrometry methods. A systematic review of the entire process at both sites was performed before the final method comparison using samples from 59 routine toxicology cases collected within a one-year time interval.

Initial differences between both laboratories were solved by focusing on important methodological aspects: (i) trueness verification of the calibration protocol focusing on the primary material, preparation of the stock solutions and adequate equilibration of calibrators and QCs, and (ii) verification of comparability of results obtained with different m/z transitions. Several of these aspects could only be verified by critically assessing spiked and native samples. After a final validation good average comparability of the two methods was observed. The average bias was -0.4%, with 85% of the differences within 20%. Moreover, the methods proved to be reproducible and robust within a one-year time interval.

This study is the first to develop a quantitative volumetric absorptive microsampling based method for PEth measurements, in addition it is the first to perform a systematic comparison of PEth measurements between two laboratories. From the discussion on the encountered pitfalls it is clear that also on a global scale, more efforts are needed to improve interlaboratory agreement.